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This Article Full Text Full Text (PDF) All Versions of this Article: 287/3/F460    most recent 00107.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thomas, C. P. Articles by Liu, K. Z. Search for Related Content PubMed PubMed Citation Articles by Thomas, C. P. Articles by Liu, K. Z.

AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5'-flanking region of the VIT32 gene

¹Department of Internal Medicine and the ²Graduate Program in Molecular Biology, University of Iowa College of Medicine, and the ³Veterans Affairs Medical Center, Iowa City, Iowa, 52246

Submitted 29 March 2004 ; accepted in final form 11 May 2004

VIT32, a vasopressin-induced transcript, inhibits Na⁺ transport when coexpressed with the epithelial sodium channel in Xenopus laevis oocytes (EMBO J 21: 5109–5117, 2002). To understand the mechanism of VIT32 gene regulation, we examined the effect of DDAVP and cAMP stimulation on VIT32 expression in M-1 mouse collecting duct cells and in H441 human airway epithelial cells. Elevation of cAMP with forskolin and IBMX increased VIT32 gene expression with a peak effect at 2 h. The increase in gene expression was abolished by H89 and by actinomycin D, suggesting that cAMP stimulates VIT32 mRNA expression by a PKA-mediated increase in gene transcription. An 1.5-kb fragment of the 5'-flanking region of VIT32 was cloned and was able to confer cAMP-stimulated reporter gene activity when transfected into M-1 and H441 cells. By deletion analysis and site-directed mutagenesis, a cAMP response element (CRE) was identified within the proximal promoter region that was sufficient to account for the increase in VIT32 gene expression seen with DDAVP and elevation of cAMP. Furthermore, DDAVP-stimulated VIT32 promoter-reporter activity was inhibited by H89 and by a dominant negative CREB construct. Finally, we were able to identify CREB as a nuclear protein that bound to the VIT32 CRE in gel mobility shift assays. In summary, DDAVP stimulates transcription of VIT32 via a CRE within the proximal promoter region of the VIT32 gene.

PP5395; epithelial sodium channel; sodium transport; airway epithelia; adenosine 3',5'-cyclic monophosphate response element binding protein